Differential detectors, comparative sensors or any other device that involves an analysis between two or more samples or specimen may result in a breakthrough peak that distracts from the measurement. A multitude of detectors or sensors are susceptible to the same or similar problems. By way of example, and without limitation, viscometers, refractive index detectors, deflection detectors, reflective detectors and any combination of refractive index, ultra-violet, fluorescent, radiochemical, electrochemical, near-infra red, mass spectroscopy, nuclear magnetic resonance, and light scattering are potential instruments, detectors and sensors applicable to the present invention.
The refractive index of a material is the most important property of any optical system that uses refraction. The refractive index is used to calculate the focusing power of lenses, and the dispersive power of prisms, and to measure the concentrations as well as elemental analyses. Since the refractive index is a fundamental physical property of a substance, it is often used to identify a particular substance, confirm its purity, or measure its concentration. The refractive index is used to measure solids, liquids, and gases. Most commonly it is used to measure the concentration of a solute in an aqueous solution. A refractometer is the instrument used to measure refractive index.
The refractive index or index of refraction of a medium is a measure for how much the speed of light, or other waves such as sound waves, is reduced inside the medium. For example, typical glass has a refractive index of 1.5, which means that light travels at 1/1.5=0.67 times the speed in air or vacuum. Two common properties of glass and other transparent materials are directly related to their refractive index. First, light rays change direction when they cross the interface from air to the material, an effect that is used in lenses and glasses. Second, light reflects partially from surfaces that have a refractive index different from that of their surroundings.
The refractive index, n, of a medium is defined as the ratio of the phase velocity c of a wave phenomenon such as light or sound in a reference medium to the phase velocity νp in the medium itself:
  n  =      c          v      p      
The refractive index, n, is most commonly used in the context of light with a vacuum as a reference medium, although historically other reference media, e.g., air at a standardized pressure and temperature, have been common. It is usually given the symbol n. In the case of light, refractive index, n, equals:n=√{square root over (εrμr)},
where εr is the material's relative permittivity, and μr is the material's relative permeability. For most materials, μr is very close to 1 at optical frequencies, therefore, n is approximately √{square root over (εr)}.
The refractive index, RI, detector is the only universal detector in high-performance liquid chromatography (HPLC). HPLC is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column.
The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell. The greater the RI difference between sample and mobile phase, the larger the imbalance will become. Thus, the sensitivity will be higher for the higher difference in RI between sample and mobile phase. On the other hand, in complex mixtures, sample components may cover a wide range of refractive index values and some may closely match that of the mobile phase, becoming invisible to the detector. The RI detector is a pure differential instrument, and any changes in the eluent composition require the rebalancing of the detector. This factor severely limits RI detector application in the analyses requiring the gradient elution, where the mobile phase composition is changed during the analysis to effect the separation. Two basic types of RI detectors are on the market today. Both require the use of a two-path cell where the sample-containing side is constantly compared with the non-sample-containing reference side.
The deflection detector is based on the deflection principle of refractometry. Refractometry provides that the deflection of a light beam is changed when the composition in a sample flow-cell changes in relation to the reference side as eluting sample moves through the system. When no sample is present in the cell, the light passing through both sides is focused on a photodetector, usually photoresistor. As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector that unbalances it. The extent of unbalance, which can be related to the sample concentration, is recorded on a recorder.
The advantages of this type of detector are: universal response; low sensitivity to dirt and air bubbles in the cells; and the ability to cover the entire refractive index range from 1.000 to 1.750 RI with a single, easily balanced cell. The disadvantages are: a general disability to easily remove and clean or replace the cell when filming or clogging occurs, the need to flush the sample-side intermittently, static solvent causing baseline drift and the need to refresh, replenish or recharge the reference-side.
Another relevant detector is a reflective detector. The reflective detector is a refractive index detector based on the Fresnel principle. In the reflective detector, the light beam is reflected from the liquid-glass interface in the detecting photocell. The introduction of sample into one cell causes light to be refracted at a different angle. The deflection of the light beam from the photoresistor causes the appearance of the electrical signal. Here, too, this difference between sample-cell signal and reference-cell signal is output to a recorder or data handling system as a peak.
The major advantage of the reflective detector is a very high sensitivity since the optics allow a higher concentration of signal in a particular RI range than is possible in other wide-range detectors. Other advantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cell accessibility, and low cost. The disadvantages of the reflective detector are the incredible sensitivity to the flow and pressure fluctuations, and the need for changing prisms to accommodate either high or low RI solvents and the need to manually adjust the optical path when making solvent changes.
The refractive index of an analyte is a function of its concentration. Change in concentration is reflected as a change in the RI. A refractive index detector for liquid chromatography should be sensitive to changes as small as 10−7 RI units corresponding to a concentration change of 1 ppm. Presence of dissolved air, changes in solvent composition, improper mixing and column bleed will contribute to baseline drift. Eluent pressure change of 15 psi will cause the change of 1×10−6 RI unit and 1° C. temperature variation will be equivalent to the change of 600×10−6 RI units. Thus it is obvious that both of these parameters must be closely controlled, especially temperature. To operate at high sensitivities, a RI detector must usually be thermostated (±0.01° C.), actually the using of the water bath connected to the detector head does not give required temperature stability, alternately, passive thermostabilisation with massive metallic block usually gives much better results.
All the above-noted apparatus, including differential detectors, comparative sensors or any device that involves an analysis between two or more samples or specimen, suffer from having a breakthrough peak that delays processing and contaminates flow paths and capillaries. Therefore, of primary concern in the present invention is the removal of the breakthrough peak, and thus, the processing delays and the contamination of the flow paths and capillaries.
It is, therefore, a feature of the present invention to remove the associated breakthrough peak.
A feature of the present invention is to improve processing efficiency by reducing processing delays.
Another feature of the present invention is to inherently prevent the contamination of the flow paths and the capillaries.
Another feature of the present invention is to reduce baseline drift by providing consistent solvent composition.
Another feature of the present invention is to reduce baseline drift by removing dissolved air from the measurement cells.
Yet another feature of the invention is to reduce baseline drift by improper mixing.
Still another feature of the present invention is to reduce baseline drift by preventing the effect of column bleed.
Another feature of the present invention is to reduce the sensitivity to the flow fluctuations.
Yet another feature of the present invention is to reduce the sensitivity to the pressure fluctuations.
Still another feature of the present invention is to easily clean the reference cell when filming or clogging occurs.
Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will become apparent from the description, or may be learned by practice of the invention. The features and advantages of the invention may be realized by means of the combinations and steps particularly pointed out in the appended claims.